The present invention is in the field of animal health, and is directed to vaccine compositions and diagnostics for disease. More particularly, the present invention relates to vaccines against mastitis in mammals, and polynucleotide molecules having nucleotide sequences encoding plasminogen activating proteins useful in the production of said vaccines.
Bovine mastitis causes significant loss of milk production in dairy cattle, resulting in severe economic impact on the dairy industry. Mastitis may be caused by one or more types of bacterial pathogens. Infection by Streptococcus is estimated to account for approximately 30% of all clinical cases of bovine mastitis. Infection by S. uberis, specifically, is estimated to account for approximately 20% of all clinical cases of bovine mastitis.
Conventional methods for the prevention and treatment of mastitis in animals include the use of sanitary milking techniques and the administration of chemical antibiotics. The use of antibiotics, however, is limited by the fact that milk containing antibiotic residues is often not considered safe for human consumption and must be discarded. Specific approaches to the treatment or prevention of mastitis in animals include that of U.S. Pat. No. 5,198,214 to Stolle et al., which describes the use of polyvalent anti-mastitis vaccines comprising inactivated mastitis-causing pathogens cultivated from the milk of infected animals. In addition, U.S. Pat. No. 5,234,684 to Sordillo et al. describes a method of treating or preventing mastitis in cows, comprising administering bovine interferon gamma to the animals.
The ability of Streptococcus spp. to infect the lactating mammary gland is dependent on the bacteria""s ability to grow in the secretion and avoid phagocytosis by bovine neutrophils (Leigh et al., 1990, Res. Vet. Sci. 49: 85-87). The majority of nitrogen in bovine milk is present in the form of protein (Aston, 1975, Austral. J. Dairy Tech. 30: 55-59) and, in the absence of proteolysis, growth of Streptococci in milk is limited by the lack of free amino acids. This is highlighted by the dependence of the lactic streptococci on extracellular, caseinolytic proteinases for growth in milk (Mills and Thomas, 1981, N. Zeal. J. Dairy Sci. Tech. 15: 43-55). In addition, the ability of bacteria to grow in milk is enhanced by the presence of the caseinolytic enzyme, plasmin (Marshall and Bramley, 1984, J. Dairy Res. 51:17-22), which breaks down milk proteins into free amino acids.
Bovine milk contains plasminogen that is normally proteolytically inactive, but which can be converted to proteolytically active plasmin by the action of a plasminogen activator. It has been demonstrated that mastitis-causing strains of Streptococcus, such as, e.g., S. uberis, produce a plasminogen activator capable of converting bovine plasminogen to plasmin. The activity of this plasminogen activator results in the break-down of milk protein to release free amino acids that, in turn, support the growth of the bacteria in the mammary gland. From this observation, it has been proposed that induction of an immune-based response, e.g., the production of neutralizing antibodies, against the plasminogen activator of Streptococcus, might serve to protect animals against mastitis. International Patent Publication WO 93/14209, which is incorporated herein by reference, demonstrates the purification of a plasminogen activator (designated therein as a xe2x80x9cstreptokinasexe2x80x9d) from culture filtrates of S. uberis, and further teaches anti-mastitis vaccine compositions based on such plasminogen activators from Streptococcus spp.
The isolation of a polynucleotide molecule having a nucleotide sequence encoding a plasminogen activator such as that, e.g., from Streptococcus, would greatly facilitate the preparation of anti-mastitis vaccines.
The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a biologically active plasminogen activator protein (designated herein as a xe2x80x9cPauA protein,xe2x80x9d previously designated as xe2x80x9cPAxe2x80x9d or xe2x80x9cstreptokinasexe2x80x9d). In a preferred embodiment, the PauA protein is from a bacterium, such as, e.g., Streptococcus, that causes or contributes to mastitis in a member of a mammalian species. In a more preferred embodiment, the species of Streptococcus is S. uberis or S. dysgalactiae. 
In a preferred embodiment, the isolated polynucleotide molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence that is the same as the amino acid sequence from about amino acid position 26 (IIe) to about amino acid position 286 (Pro) of SEQ ID NO:2 or SEQ ID NO:4. In a non-limiting embodiment, such polynucleotide molecule comprises the nucleotide sequence from about nucleotide position 195 to about nucleotide position 977 of SEQ ID NO:1, or from about nucleotide position 196 to about nucleotide position 978 of SEQ ID NO:3, respectively.
In a further preferred embodiment, the isolated polynucleotide molecule of the present invention comprises a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence from about amino acid position 1 (Met) to about amino acid position 286 (Pro) of SEQ ID NO:2 or SEQ ID NO:4. In a non-limiting embodiment, such polynucleotide molecule comprises the nucleotide sequence from about nucleotide position 120 to about nucleotide position 980 of SEQ ID NO:1, or from about nucleotide position 121 to about nucleotide position 981 of SEQ ID NO:3, respectively. In a further non-limiting embodiment, the polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:1, or the nucleotide sequence of SEQ ID NO:3, respectively.
The present invention further provides a polynucleotide molecule that is substantially homologous to the polynucleotide molecule of the present invention encoding a PauA protein.
The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is substantially homologous to a PauA protein encoded by a polynucleotide molecule of the present invention.
The present invention further provides a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment of a PauA protein or substantially homologous polypeptide of the present invention. In a specific though non-limiting embodiment, the present invention provides a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment consisting of a sub-sequence of amino acids from SEQ ID NO:2 or SEQ ID NO:4 selected from the group consisting of amino acids at positions of about 28 to about 286, about 104 to about 286, about 172 to about 286, about 28 to about 170, about 104 to about 170, about 104 to about 208, about 172 to about 208, about 28 to about 103, about 28 to about 208, and about 149 to about 286.
The present invention further provides a polynucleotide molecule comprising a nucleotide sequence that encodes a fusion protein comprising a PauA protein, substantially homologous polypeptide, or peptide fragment of the present invention joined to a carrier or fusion partner.
The present invention further provides oligonucleotide molecules that are useful as primers in amplification techniques, and as diagnostic probes in differential disease diagnosis. In a non-limiting embodiment, an oligonucleotide molecule of the present invention consists of a nucleotide sequence selected from the group consisting of SEQ ID NOS:13-27 and the complements of said sequences. In a further non-limiting embodiment, an oligonucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:13-27 and the complements of said sequences.
The present invention further provides compositions and methods for the recombinant expression of a polynucleotide molecule of the present invention, including recombinant cloning vectors and recombinant expression vectors comprising the polynucleotide molecule, and host cell lines transformed with any of said vectors.
The present invention further provides a recombinantly-expressed PauA protein, substantially homologous polypeptide, peptide fragment, or fusion protein encoded by a polynucleotide molecule of the present invention.
The present invention further provides analogs and derivatives of a PauA protein, substantially homologous polypeptide, peptide fragment or fusion protein of the present invention.
The present invention further provides a vaccine for protecting a member of a mammalian species against mastitis, comprising an immunologically effective amount of a PauA protein, substantially homologous polypeptide, peptide fragment, fusion protein, analog, derivative, or polynucleotide molecule of the present invention that is capable of inducing a protective response against mastitis in the mammal, and a veterinarily acceptable carrier. The vaccine can optionally comprise an adjuvant or other immunomodulatory component.
In a non-limiting embodiment, the vaccine of the present invention is a combination vaccine for protecting a member of a mammalian species against mastitis and, optionally, one or more other diseases or pathological conditions that can afflict the mammal, which combination vaccine comprises an immunologically effective amount of a first component comprising a PauA protein, substantially homologous polypeptide, peptide fragment, fusion protein, analog, derivative, or polynucleotide molecule of the present invention that is capable of inducing a protective response against mastitis in the mammal; an immunologically effective amount of a second component comprising an antigen capable of inducing a protective response against a disease or pathological condition that can afflict the mammal; and a veterinarily acceptable carrier.
The present invention further provides a method of preparing a vaccine for protecting a member of a mammalian species against mastitis, comprising combining an immunologically effective amount of a PauA protein, substantially homologous polypeptide, peptide fragment, fusion protein, analog, derivative, or polynucleotide molecule of the present invention that is capable of inducing a protective response against mastitis in the mammal, with a veterinarily acceptable carrier.
The present invention further provides a method of vaccinating a member of a mammalian species against mastitis, comprising administering the vaccine of the present invention to the mammal.
The present invention further provides antibodies that specifically bind to a PauA protein, substantially homologous polypeptide, peptide fragment, fusion protein, analog, or derivative of the present invention.
The present invention further provides vaccine kits and diagnostic kits.